The regulation of oxygen (O2) supply to match demand in skeletal muscle is such a fundamental, physiological process that it is often assumed that the mechanisms responsible are well understood. However, although numerous theories have been proposed, none has been adequately tested in vivo. This is not surprising given the complexity of the microvascular regulatory systems that respond to O2 as well as the complexity of O2 transport where O2 supply is determined by flow distribution, diffusional O2 exchange among all vessels and rheological properties of erythrocytes (RBCs) flowing in bifurcating networks. Unraveling the complexity of this biological system requires a systems biology approach in which experiments provide information on the things we can determine and mathematical computation using that experimental evidence enables us to predict those factors which elude us. Although models of O2 delivery have existed since the time of August Krogh, few have incorporated the necessary regulatory component since its identity has remained elusive. Recent studies have supported a role for the O2 carrying RBC as an important regulatory component that alters O2 supply to meet demand via the release of adenosine 5'-triphosphate (ATP). In the microcirculation, ATP released from RBCs in response to reduced O2 tension in capillaries or venules can function in a paracrine fashion to produce vasodilation locally as well as vasodilation that is conducted to upstream arterioles. RBC-derived ATP can also function in an autocrine fashion to stimulate the release of vasodilator epoxyeicosatrienoic acids (EETs) from RBCs. The goal of this project is to substantiate the growing evidence for a critical role for RBCs in the regulation of the matching of O2 supply with need in skeletal muscle. This will be accomplished using a new dynamic computational O2 transport model which is based on experimental data integrating the geometrical complexity of the microvasculature and surrounding tissue with a network model of microvascular flow as well as a convective and diffusive O2 transport model within a 3D tissue volume. This proposal will determine whether O2-saturation dependent release of ATP from RBCs is responsible for the local regulation of O2 supply within skeletal muscle. The O2 regulatory model will be developed, tested and refined in stages beginning with the existing experiment-based model and development of an empirical algorithm which simulates the RBC hemodynamic and O2 saturation response observed in experiments. Concomitantly, quantitative and temporal data on the release of ATP and EETs from RBCs exposed to reduced O2 and their vasoactivity in the rat microcirculation will be collected and used to refine the model, ultimately replacing the empirical algorithm. A defect in ATP release from RBCs of diabetic animals will be used to challenge the regulatory model. Substituting experimental data from a rat model of diabetes for data obtained in their matched controls in the computational model will provide important new information on the importance of RBC-derived ATP in the defect in skeletal muscle microcirculation associated with diabetes. The regulation of oxygen supply to match oxygen demand in skeletal muscle is a fundamental physiological process, yet because of its complexity, attempts to describe it have been generally inadequate. It has become increasingly obvious that because processes like these cannot be understood merely by reducing them to their component parts, they must be studied as intact, functioning systems using a systems biology approach with computational modeling. In this proposal we use a systems biology approach to determine whether the release of ATP from red blood cells in response to metabolic need is responsible for local regulation of oxygen supply within skeletal muscle and test the predictions of the model by examining it using a system (type 2 diabetes) in which there is a defect in that regulatory system, i.e., ATP release from RBCs of type 2 diabetics is compromised.